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1 Supplementary Material and Figures Nutrient transport suggests an evolutionary basis for charged archaeal surface layer proteins Po-Nan Li et al. Supplementary Figure S1. Numerical convergence of the simulation. We monitored the difference of the numerical solution of the concentration at the active site between two consecutive time steps, and observed an error rate less than 10-4 within 1,000 time steps. (A) The evolutions of NH 4 concentration at the active site over simulation time; the plot shows that the simulation converges to a steady-state. (B) The error, defined as, is monotonically decreasing over time. The local minimum around 7000 steps reflects asymmetric convergence of the ion species (Fig S4B).
2 Supplementary Figure S2. Comparative NH 4 concentration profiles in the simulation domain with a negatively, neutral, and positively charged S-layer. (A) NH 4 concentration profile with a positively charged S-layer, resulting in sharply lower NH 4 concentration in the PPS. (B) NH 4 concentration profiles for neutral and charged S-layers across the length of the simulation domain at the x-location of the active site. NH 4 concentrations in the PPS with a positively charged S-layer are an order of magnitude lower than those with a negative S-layer. (C) NH 4 concentration at the active site as a function of the surface charge density of the S-layer. The black data point indicates no surface charge. 1
3 Supplementary Figure S3 Concentration profiles of NH 3 and NH 4 throughout the simulation domain for a neutral and negatively charged S-layer at steady state. The concentration of NH 4 is not affected by a neutral S-layer (black line), resulting in a reduced concentration at the active site compared to a charged S-layer (orange line). The concentration of NH 3, which is not charged, is not modulated by a charged S-layer. At ph = 7.5, NH 4 dominates total ammonia (NH 3 NH 4 ) bulk concentration, with corresponding NH 3 bulk concentration of just 0.2 nm. Active site NH 3 concentration is sharply reduced to 24 pm (brown and pink lines). Supplementary Figure S4. Fluxes (J y [M m -2 μs -1 ]) of NH4 (orange), Cl- (blue), and Na (red) through the nanopore as a function of time. Fluxes were measured in the middle of the channel. A) For a neutral S-layer, co- and counterion fluxes converge to finite values at steady state. B) For a charged S-layer, co- and counterion fluxes converged exponentially fast. 2
4 Supplementary Figure S5. Boundary conditions applied in the simulation. NH 4 ions reacted at the active site are compensated by adding the same amount of ions to the Dirichlet boundary condition at the top boundary. Supplementary Figure S6 Hexagonal arrangement of S-layer proteins. Each orange rhombus represents a single S-layer protein monomer, and each grey triangle represents a monomer shared with a neighboring hexagonal tile. 3
5 Supplementary Table S1. Accession numbers of archaeal S-layer gene sequences studied in this work. As most archaeal S-layer genes have not been positively identified, we selected SLP sequences based on genome annotations; however, all organisms represented have previously been observed to possess an S-layer (Albers and Meyer, 2011). The list includes virtually all known archaeal clades. Organism Nitrosopumilus maritimus Nitrosoarchaeum limnia Korarchaeum cryptofilum Thermoproteus tenax Pyrobaculum ferrireducens Acidianus ambivalens Metallosphaera sedula Sulfolobus solfataricus Desulfurococcus amylolyticus Staphylothermus marinus Pyrococcus furiosus Methanothermus fervidus Methanothermus sociabilis Methanocaldococcus jannaschii Methanococcus maripaludis Ferroglobus placidus Archaeoglobus fulgidus Picrophilus torridus Halobacterium salinarum Haloferax volcanii Haloquadratum walsbyi Methanosarcina acetivorans Methanosarcina mazei Methanospirillum hungatei Methanoculleus marisnigri NCBI Accession ABX13097 EGG41144 WP_ WP_ AET32696 CAM84437 ABP94767 WP_ ACL10820 AAC44118 WP_ ADP77870 P27374 WP_ ABR66257 ADC66245 WP_ WP_ AAA72185 WP_ CCC39200 WP_ CAA54899 WP_ ABN
6 Supplementary Table S2. Total potential (V) in Volts over the nanopore, current (I) in Amperes, and conductance (G) in Siemens for a neutral and charged S-layer for each of the ion species. NH4 only Cl - only Na Only Combined NH4 conc. V I G I G I G I G 10 nm 6.7E E E E E E E E E-19 Neutral 100 nm 7.1E E E E E E E E E nm 6.7E E E E E E E E E nm -2.4E E E E E E E E E-17 Charged 100 nm -2.7E E E E E E E E E nm -2.8E E E E E E E E E-18 5
7 Supplementary Methods SLP glycosylation by mass spectrometry. Cell lysate from cultures in late exponential growth was collected after mechanical lysis in a urea lysis buffer (9 M urea; 20 mm HEPES, ph 8.0). Lysate was submitted for proteomic analysis at the Vincent Coates Foundation Mass Spectrometry Laboratory, Stanford University Mass Spectrometry ( Briefly, proteins were crashed with acetone, centrifuged, and the protein pellet dried, reduced, and alkylated. Following a trypsin digest, peptides were column-purified and injected onto a Waters NanoAquity Ultra Performance Liquid Chromatography instrument. The mass spectrometer was a Orbitrap Elite, and resulting data was searched using Byonic (Protein Metrics) and processed using Matlab and Excel to display proteins and the number of peptides associated with them. N-linked glycosylation searches were performed with 2D posterior error probability (PEP 2D) cutoff of Supplementary Note. In the neutral and charged S-layer case, the point-like ion sink performs work to move NH 4 ions across the nanopore, increasing their electrical potential. The flux of positive charges creates a negatively (upward) oriented electric field. In turn, Na and Cl - migrate along their (small) electrical-chemical gradients in the pore. The redistribution of electrical charge and chemical potential creates fluxes of Na and Cl - ions over the domain boundary. An infinitesimally small flux of co- and counterions will persist. The effect of excess or lack of co- and counterions accumulates around the point-like ion sink. 6
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